Rapid evaluation of peat-base legume inoculant using immunomagnetic beads for cell retrieval and fluorescent nucleic acid probes for viability analysis

Problems associated with traditional methods of evaluating legume inoculants using the most-probable-number plant nodule grow-out test include technician time, expense, growth chamber space, and the length of time (30 days) required to conduct the tests. Through the use of specific monoclonal antibodies, a biotin-labelled intermediate antibody, and streptavidin-labelled magnetic micro-beads, we were able to rapidly remove and purify rhizobial cells from peat inoculants used as quality control samples in the Canadian Legume Inoculant and Pre-Inoculated Seed Testing Program. Viability of the recovered cells was evaluated with a commercial kit employing differential fluorescent nucleic acid probes allowing viability to be rapidly determined via visual examination with a fluorescence microscope. Total elapsed time for complete evaluation of an inoculant was about 90 minutes.     

Limitations of the enzyme-linked immunosorbent assay for routine determination of legume inoculant quality

Peat inoculants containing strains of either Rhizobium or Bradyrhizobium spp. were used to determine correlations between cell numbers and A₄₀₅ values obtained with double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and indirect ELISA. ELISA values of inoculants containing strains of Rhizobium were weak and non-specific; with Bradyrhizobium spp. strains, readings were higher and cross-reactions negligible when heated inoculant suspensions were allowed to stand for 3 h before ELISA determinations were made. With soybean inoculant, correlation coefficients of r = 0.93 and 0.83 were obtained with DAS and indirect ELISA, respectively. A linear curve relating log cell numbers to A₄₀₅ values was used to determine the reliability of DAS ELISA values obtained over 2 years with tests on commercially produced soybean inoculants. In the range 5 times 10⁸-ca 3 times 10⁹ cells/g inoculant, DAS ELISA estimates closely followed plate counts but no significant correlation was found when inoculants contained >ca 3 times 10⁹ cells/g. With a minimum requirement of 1 times 10⁹ cells/g inoculant, discrepancies between DAS ELISA estimates and plate counts obtained with inoculants produced with gamma-irradiated peat would have resulted in the erroneous rejection or acceptance of 14.5% of all inoculants tested, based on DAS ELISA estimates. With inoculants produced with steam-sterilized peat, which was unfavourable for survival of strain WB1, 70.0% of the inoculants rejected because of low plate counts would have been acceptable on the basis of DAS ELISA estimates.     

Mineral Soils as Carriers for Rhizobium Inoculants

Mineral soil-based inoculants of Rhizobium meliloti and Rhizobium phaseoli survived better at 4°C than at higher temperatures, but ca. 15% of the cells were viable at 37°C after 27 days. Soil-based inoculants of R. meliloti, R. phaseoli, Rhizobium japonicum, and a cowpea Rhizobium sp. applied to seeds of their host legumes also survived better at low temperatures, but the percent survival of such inoculants was higher than peat-based inoculants at 35°C. Survival of R. phaseoli, R. japonicum, and cowpea rhizobia was not markedly improved when the cells were suspended in sugar solutions before drying them in soil. Nodulation was abundant on Phaseolus vulgaris derived from seeds that had been coated with a soil-based inoculant and stored for 165 days at 25°C. The increase in yield and nitrogen content of Phaseolus angularis grown in the greenhouse was the same with soil-and peat-based inoculants. We suggest that certain mineral soils can be useful and readily available carriers for legume inoculants containing desiccation-resistant Rhizobium strains.     

Genetic diversity and symbiotic effectiveness of Phaseolus vulgaris-nodulating rhizobia in Kenya

Phaseolus vulgaris (common bean) was introduced to Kenya several centuries ago but the rhizobia that nodulate it in the country remain poorly characterised. To address this gap in knowledge, 178 isolates recovered from the root nodules of P. vulgaris cultivated in Kenya were genotyped stepwise by the analysis of genomic DNA fingerprints, PCR-RFLP and 16S rRNA, atpD, recA and nodC gene sequences. Results indicated that P. vulgaris in Kenya is nodulated by at least six Rhizobium genospecies, with most of the isolates belonging to Rhizobium phaseoli and a possibly novel Rhizobium species. Infrequently, isolates belonged to Rhizobium paranaense, Rhizobium leucaenae, Rhizobium sophoriradicis and Rhizobium aegyptiacum. Despite considerable core-gene heterogeneity among the isolates, only four nodC gene alleles were observed indicating conservation within this gene. Testing of the capacity of the isolates to fix nitrogen (N₂) in symbiosis with P. vulgaris revealed wide variations in effectiveness, with ten isolates comparable to Rhizobium tropici CIAT 899, a commercial inoculant strain for P. vulgaris. In addition to unveiling effective native rhizobial strains with potential as inoculants in Kenya, this study demonstrated that Kenyan soils harbour diverse P. vulgaris-nodulating rhizobia, some of which formed phylogenetic clusters distinct from known lineages. The native rhizobia differed by site, suggesting that field inoculation of P. vulgaris may need to be locally optimised.     

根瘤菌菌剂的研究与开发现状

根瘤菌与豆科植物共生成为豆科植物固氮的重要方式,它可以为豆科植物提供所需氮量的1/2～1/3。因此,土壤中有效根瘤菌的数量是决定豆科植物产量的重要因素,而根瘤菌菌剂的使用可以有效地提高土壤中根瘤菌数量。本文从根瘤菌菌剂制备中高效菌种的选育及匹配、高密度菌剂的制备、菌剂保存方法等方面进行综述。比较了自然选育、杂交选育和诱变选育等各类选育方法及琼脂试管配对法和水培配对法的优缺点;总结了菌剂制备的一般过程和方法;论述了菌剂保藏过程中冷冻干燥法和各种保护剂的使用对菌剂保藏效果的影响。本文阐述了根瘤菌菌剂的制备工艺和发展方向,为根瘤菌剂的研制提供重要参考。     

Dilution of Liquid Rhizobium Cultures To Increase Production Capacity of Inoculant Plants

Experiments were undertaken to test whether peat-based legume seed inoculants, which are prepared with liquid cultures that have been deliberately diluted, can attain and sustain acceptable numbers of viable rhizobia. Liquid cultures of Rhizobium japonicum and Rhizobium phaseoli were diluted to give 10⁸, 10⁷, or 10⁶ cells per ml, using either deionized water, quarter-strength yeast-mannitol broth, yeast-sucrose broth, or yeast-water. The variously diluted cultures were incorporated into gamma-irradiated peat, and the numbers of viable rhizobia were determined at intervals. In all of the inoculant formulations, the numbers of rhizobia reached similarly high ceiling values by 1 week after incorporation, irrespective not only of the number of cells added initially but also of the nature of the diluent. During week 1 of growth, similar multiplication patterns of the diluted liquid cultures were observed in two different peats. Numbers of rhizobia surviving in the various inoculant formulations were not markedly different after 6 months of storage at 28°C. The method of inoculant preparation did not affect the nitrogen fixation effectiveness of the Rhizobium strains.     

Survival of Rhizobium phaseoli in Coal-Based Legume Inoculants

The long-term survival of Rhizobium phaseoli strains 127K17, 127K26, and 127K35 in legume inoculants prepared with eight different coals (one strain and one coal per inoculant) was studied. The coals used were Pennsylvania anthracite, bituminous coals from Illinois, Pennsylvania, and Utah, lignite from North Dakota and Texas, and subbituminous coals from New Mexico and Wyoming; they ranged in pH from 4.7 to 7.5 All coals, with the exceptions of Illinois bituminous coal and Texas lignite (pH's of 5.0 and 4.7, respectively), supported the growth and survival of all R. phaseoli strains. All coal-based inoculants in which rhizobial viability was maintained had more than 10⁶ rhizobia per g for at least 7 months, and most contained more than 10⁷ rhizobia per g after 12 months. It appears that most coals, regardless of grade or source, may be acceptable carriers for R. phaseoli inoculants.     

Rhizobium Strain Identification and Quantification in Commercial Inoculants by Immunoblot Analysis

An immunoblot procedure for the strain-specific quantitative analysis of commercial Rhizobium inoculants was developed. The technique greatly reduced the time required for inoculant analysis. Correlation between immunoblot analysis and traditional plant nodule grow-out most-probable-number techniques was r = 0.90 for 16 commercial alfalfa inoculants tested.     

Rhizobium inoculant for soybean [Glycine max (L.) Merrill] in Mekong Delta

Summary Rhizobial inoculation trials were conducted in an acid heavy clay soil in Mekong Delta, Viet Nam, using peat based inoculants produced locally and the commercial granular product of Nitragin CCo., Wisconsin, USA. The pH of these soils ranged from 4.5 to 5.1. Two soybean cultivars, MTD₆ and MTD₁₀, were tested as host plants. There were no significant differences between locally made inoculant treated plants and the uninoculated controls in both cultivars. But, the Nitragin inoculation improved all plant characteristics examined in both cultivars. Grain yields of Nitragin inoculated plants of cultivar MTD₆ and cultivar MTD₁₀ were 6.5 and 5.5 times as much as those of the controls; protein content of grain increased 11 and 16 percent, respectively. Well nodulated plants had shorter life cycles, flowering durations, and days to flowering. The Rhizobium symbiosis resulted in an additional 153 kg grain-N/ha. These studies show that a surface coated commercial multistrain inoculant can be used to successfully grow soybeans in the acid, heavy clay soils of the Mekong Delta.     

Semienclosed Tube Cultures of Bean Plants (Phaseolus vulgaris L.) for Enumeration of Rhizobium phaseoli by the Most-Probable-Number Technique

The semienclosed tube culture technique of Gibson was modified to permit growth of common bean (Phaseolus vulgaris L.) roots in humid air, enabling enumeration of the homologous (nodule forming) symbiont, Rhizobium phaseoli, by the most-probable-number plant infection method. A bean genotype with improved nodulation characteristics was used as the plant host. This method of enumeration was accurate when tubes were scored 3 weeks after inoculation with several R. phaseoli strains diluted from aqueous suspensions, peat-based inoculants, or soil. A comparison of population sizes obtained by most-probable-number tube cultures and plate counts indicated that 1 to 3 viable cells of R. phaseoli were a sufficient inoculant to induce nodule formation.     

Effectiveness of Rhizobium Strains Used in Inoculants after Their Introduction into Soil

Rhizobium strains used in inoculants for Trifolium spp., Medicago spp., Glycine max, and Lotus pedunculatus were isolated from nodules of these legumes grown in soils into which the rhizobia had been introduced 4 to 8 years before. Isolations were made from a total of 420 nodules. Nodule occupancy by the inoculant strains varied from 17.7% for a soybean strain to 100% in the case of L. pedunculatus whose specific rhizobia did not occur in the soils studied. In general, inoculant strains isolated from nodules did not differ in effectiveness from cultures of the same strains concurrently maintained in lyophilized form. The average effectiveness of all of the isolates (identified and unidentified) from a legume was 7.1 to 73.3% higher than that of the unidentified isolates alone, demonstrating the prolonged effect that a single-seed inoculation has on the rhizobial population in a soil which had not been planted with legumes before. Relatively weak recovery of a Rhizobium japonicum strain introduced into soil 4 years after soybean seed inoculated with a different strain had been planted in the same soil confirmed the advantage of a resident population over an introduced inoculant strain.     

Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus

The need for sustainable agricultural practices is revitalizing the interest in biological nitrogen fixation and rhizobia-legumes symbioses, particularly those involving economically important legume crops in terms of food and forage. The genus Mesorhizobium includes species with high geographical dispersion and able to nodulate a wide variety of legumes, including important crop species, like chickpea or biserrula. Some cases of legume-mesorhizobia inoculant introduction represent exceptional opportunities to study the rhizobia genomes evolution and the evolutionary relationships among species. Complete genome sequences revealed that mesorhizobia typically harbour chromosomal symbiosis islands. The phylogenies of symbiosis genes, such as nodC, are not congruent with the phylogenies based on core genes, reflecting rhizobial host range, rather than species affiliation. This agrees with studies showing that Mesorhizobium species are able to exchange symbiosis genes through lateral transfer of chromosomal symbiosis islands, thus acquiring the ability to nodulate new hosts. Phylogenetic analyses of the Mesorhizobium genus based on core and accessory genes reveal complex evolutionary relationships and a high genomic plasticity, rendering the Mesorhizobium genus as a good model to investigate rhizobia genome evolution and adaptation to different host plants. Further investigation of symbiosis genes as well as stress response genes will certainly contribute to understand mesorhizobia-legume symbiosis and to develop more effective mesorhizobia inoculants.     

Rhizobial counts in peat inoculants vary amongst legume inoculant groups at manufacture and with storage: implications for quality standards

Background and aims

Inoculation of legumes at sowing with rhizobia has arguably been one of the most cost-effective practices in modern agriculture. Critical aspects of inoculant quality are rhizobial counts at manufacture/registration and shelf (product) life.

Methods

In order to re-evaluate the Australian standards for peat-based inoculants, we assessed numbers of rhizobia (rhizobial counts) and presence of contaminants in 1,234 individual packets of peat–based inoculants from 13 different inoculant groups that were either freshly manufactured or had been stored at 4 °C for up to 38 months to determine (a) rates of decline of rhizobial populations, and (b) effects of presence of contaminants on rhizobial populations. We also assessed effects of inoculant age on survival of the rhizobia during and immediately after inoculation of polyethylene beads.

Results

Rhizobial populations in the peat inoculants at manufacture and decline rates varied substantially amongst the 13 inoculant groups. The most stable were Sinorhizobium, Bradyrhizobium and Mesorhizobium with Rhizobium, particularly R. leguminosarum bv. trifolii the least stable. The presence of contaminants at the 10^?6 level of dilution, i.e. >log 6.7 g^?1 peat, reduced rhizobial numbers in the stored inoculants by an average of 37 %. Survival on beads following inoculation improved 2–3 fold with increasing age of inoculant.

Conclusions

We concluded that the Australian standards for peat-based rhizobial inoculants should be reassessed to account for the large differences amongst the groups in counts at manufacture and survival rates during storage. Key recommendations are to increase expiry counts from log 8.0 to log 8.7 rhizobia g^?1 peat and to have four levels of inoculant shelf life ranging from 12 months to 3 years.     

Inoculant Production with Diluted Liquid Cultures of Rhizobium spp. and Autoclaved Peat: Evaluation of Diluents, Rhizobium spp., Peats, Sterility Requirements, Storage, and Plant Effectiveness

Fully grown broth cultures of various fast- and slow-growing rhizobia were deliberately diluted with various diluents before their aseptic incorporation into autoclaved peat in polypropylene bags (aseptic method) or mixed with the peat autoclaved in trays (tray method). In a factorial experiment with the aseptic method, autoclaved and irradiated peat samples from five countries were used to prepare inoculants with water-diluted cultures of three Rhizobium spp. When distilled water was used as the diluent, the multiplication and survival of rhizobia in the peat was similar to that with diluents having a high nutrient status when the aseptic method was used. In the factorial experiment, the mean viable counts per gram of inoculant were log 9.23 (strain TAL 102) > log 8.92 (strain TAL 82) > log 7.89 (strain TAL 182) after 24 weeks of storage at 28°C. The peat from Argentina was the most superior for the three Rhizobium spp., with a mean viable count of log 9.0 per g at the end of the storage period. The quality of inoculants produced with diluted cultures was significantly (P = 0.05) better with irradiated than with autoclaved peat, as shown from the factorial experiment. With the tray method, rhizobia in cultures diluted 1,000-fold or less multiplied and stored satisfactorily in the presence of postinoculation contaminants, as determined by plate counts, membrane filter immunofluorescence, and plant infection procedures. All strains of rhizobia used in both the methods showed various degrees of population decline in the inoculants when stored at 28°C. Fast- and slow-growing rhizobia in matured inoculants produced by the two methods showed significant (P < 0.01) decline in viability when stored at 4°C, whereas the viability of some strains increased significantly (P < 0.01) at the same temperature. The plant effectiveness of inoculants produced with diluted cultures and autoclaved peat did not differ significantly from that of inoculants produced with undiluted cultures and gamma-irradiated peat.     

Identification of Rhizobium spp. in peat-based inoculants by DNA hybridization and PCR and its application in inoculant quality control.

Procedures based on DNA hybridization and PCR were developed for quality control of Rhizobium inoculants. Inoculants for pea and goat's rue were produced by Elomestari Ltd., Juva, Finland, in sterile dry fine peat by the standard procedure used by the company. The inoculants contained Rhizobium galegae HAMBI 1174 and HAMBI 1207 and an R. leguminosarum biovar vicia strain, 16HSA, either solely or in combinations of two or three strains. DNA was isolated from 1-g samples of each peat inoculant and analyzed by nonradioactive DNA-DNA hybridization and by PCR. The hybridization probes were total DNAs from pure cultures of R. galegae HAMBI 1207 and R. leguminosarum biovar viciae 16HSA and a 264-bp strain-specific fragment from the genome of R. galegae HAMBI 1174. The total DNA probes distinguished inoculants containing R. galegae or R. leguminosarum, and the strain-specific probe distinguished inoculants containing R. galegae HAMBI 1174. The hybridization results for R. galegae were verified in a PCR experiment by amplifying an R. galegae species-specific fragment and an R. galegae HAMBI 1174 strain-specific fragment in the same reaction. When suitable probes and primers are available, the methods described here offer promising alternatives for the quality control of peat-based inoculants.     

Inoculant Maturity Influences Survival of Rhizobia on Seed

Survival of Rhizobium trifolii on seeds of arrowleaf clover (Trifolium versiculosum Savi) and subclover (Trifolium subterraneum L.) was affected by the maturity of peat-, vermiculite-, and charcoal-based inoculants. Ten times more rhizobia survived on seed 4 days after inoculation when inoculants were stored (cured) before being utilized as compared with uncured inoculants. Increasing the curing time of inoculants beyond 4 weeks had little effect on increasing survival of seed-applied rhizobia.     

Polymer-entrapped rhizobium as an inoculant for legumes

Summary Field and cylinder experiments conducted in France and in Senegal showed that polyacrylamide, previously proposed as an entrapping gel for preparing Rhizobium inoculants, could be replaced by alginate (AER inoculant) or a mixture of xanthan and carob gum (XER inoculant). Semi-dried or dried AER and XER were used successfully provided that their storage time was less than 90 days. In soil inoculation trails, no marked differences were observed among semi-dried XER, dried AER, and dried XER. A number of seed inoculation experiments indicated that dried XER significantly outranked AER. Seeds preinoculated by up to 48 days with XER yielded plants which were comparable in nodulation and growth parameters to those derived from plant receiving peat inoculation at the time of planting.     

Improvement of Rhizobium Inoculants

A practical approach was used to develop a Rhizobium (Bradyrhizobium) japonicum inoculant that increases soybean (Glycine max (L.) Merr.) yield in fields with indigenous Rhizobium populations, which typically outcompete strains present in existing commercial inoculants and therefore decrease the value of inoculant use. Field tests managed by several universities in the Mississippi delta region averaged a 169-kg/ha (P < 0.01) grain yield increase. The inoculant contains a mixture of mutants selected for increased nitrogen fixation ability. These mutants were derived from indigenous wild-type strains that are capable of high-level occupancy of nodules in soybean fields in the Mississippi delta region. To ensure microbiological purity, the inoculant is fermented directly in the point-of-use container with a vermiculite carrier (L. Graham-Weiss, M. L. Bennett, and A. S. Paau, Appl. Environ. Microbiol. 53:2138-2140, 1987). It should be possible to use this approach to produce more effective Rhizobium inoculants for any legume in any geographical area.     

Single-Strain versus Multistrain Inoculation: Effect of Soil Mineral N Availability on Rhizobial Strain Effectiveness and Competition for Nodulation on Chick-Pea, Soybean, and Dry Bean

The nitrogen-fixing effectiveness of multistrain inoculants was found to be determined by both the effectiveness of the component strains and the percentage of the nodules occupied by them. Multistrain formulations were always either as good as the most effective single-strain inoculant or intermediate between the most and the least effective. The percentage of nodules occupied and the amount of nitrogen fixed by the component strains of a multistrain inoculant showed highly significant linear correlation. The availability of soil N had a significant influence on the nitrogen fixation potential of each strain. The mineral N status of the soil was clearly a significant factor in affecting the competition pattern of Rhizobium loti (chick-pea) and Bradyrhizobium japonicum strains. Differences between the effectiveness of strains were masked under conditions of soil N availability. However, when soil N was immobilized with sugarcane bagasse, the differences became significant. In the chick-pea system, R. loti TAL 1148 (Nit 27A8) was the most effective but not the most competitive of the three strains used. In the soybean and dry bean systems, B. japonicum TAL 102 (USDA 110) and R. leguminosarum bv. phaseoli TAL 182, respectively, were consistently the most effective and, more often than not, the most competitive of the strains used for each species.     

Enhancing the Efficiency of Soybean Inoculant for Nodulation under Multi-Environmental Stress Conditions

The development of rhizobial inoculants with increased resistance to abiotic stress is critical to mitigating the challenges related to climate change. This study aims at developing a soybean stress-tolerant Bradyrhizobium inoculant to be used under the mixed stress conditions of acidity, high temperature, and drought. Six isolates of Bradyrhizobium with high symbiotic performance on soybean were tested to determine their growth or survival abilities under in vitro conditions. The representative stress-tolerant Bradyrhizobium isolates 184, 188, and 194 were selected to test their ability to promote soybean growth under stress conditions compared to the type strain Bradyrhizobium diazoefficiens USDA110. The plant experiment indicated that isolate 194 performed better in symbiosis with soybean than other Bradyrhizobium strains under stress conditions. Based on the stress tolerance index, soybeans inoculated with isolate 194 showed a high growth performance and significantly better nodulation competition ability than USDA110 under several stress conditions. Interestingly, supplementation of sucrose in the culture medium significantly enhances the survival of the isolate and leads to improved plant biomass under various stress conditions. Analysis of the intra-cellular sugars of isolate 194 supplemented with sucrose showed the accumulation of compatible solutes, such as trehalose and glycerol, that may act as osmoprotectants. This study indicates that inoculation of stress-tolerant Bradyrhizobium together with sucrose supplementation in a medium could enhance bacterial survival and symbiosis efficiency under stress conditions. Although it can be applied for inoculant production, this strategy requires validation of its performance in field conditions before adopting this technology.